PROJECT SUMMARY Gammaherpesviruses (GHVs) such as Epstein-Barr virus (EBV) and Kaposi sarcoma-associated herpesvirus (KSHV) persist for the life of the infected host and place the host at risk for numerous cancers. Vaccines for EBV and KSHV are not available, and there is no consensus strategy for vaccine development. Studies using murine gammaherpesvirus 68 (MHV68), a highly tractable mouse model of GHV infection and disease, suggest that vaccination against GHVs has the capacity to reduce or eliminate morbidity and mortality due to GHV infection. The major goal of this proposal is to evaluate the efficacy of novel mutant-virus vaccine approaches using the MHV68 model of GHV pathogenesis. A critical barrier to using replication-dead or defective viruses as vaccine platforms is the reversion of the mutant virus back to original wild-type (WT) sequence. To address this major obstacle to safe vaccine production, we developed a complementation technique that eliminates WT reversion. We have successfully utilized this approach to generate high-titer, replication-dead GHV stocks in which the gene encoding a viral lytic transactivator protein, RTA, was disrupted. Experiments proposed here will apply this technique to develop candidate MHV68 vaccine strains and define parameters of immune protection in mice. We will forward-engineer viruses to disrupt two classes of GHV genes: (1) genes of the immediate-early, early, or late gene classes that are essential for full lytic cycle progression and (2) determinants of latency that are necessary for oncogenesis. Safety of vaccine candidates will be tested in immunodeficient mice. We will next apply established platforms in the MHV68 pathogenesis system to test our hypothesis that replication-dead viruses will generate immune responses that reduce viral burden and prevent GHV infection. These experiments use an established animal model of GHV infection and disease to provide a preclinical test of the utility of therapeutic and/or protective vaccines for EBV and KSHV. Data generated through this proposal ultimately will impact human health by providing a safe and effective approach to the development of GHV-specific vaccines to limit GHV-associated cancers and provide a road-map for the development of vaccines targeting other HVs, such as herpes simplex virus and human cytomegalovirus.